This invention relates to peptide-based compounds having light-emitting moieties. Peptides may be chemically linked with detectable xe2x80x9clabelsxe2x80x9d and used as probes, for example, to monitor peptide, cytokine, drug, and hormone receptors at the cellular level. Typically, the labeled peptide is placed in contact with a tissue or cell culture where it binds to an available receptor. Once bound, the label is detected, allowing properties such as receptor distribution or receptor binding kinetics to be monitored.
Peptides are typically labeled with radioactive elements such as 125I or 3H. In this case, emission of high-energy radioactive particles is monitored using standard xcex3-ray detectors, thereby allowing detection of the label. While detection techniques for 125I and 3H are well-known, radioactive compounds by nature have limited half lives, and are often both toxic and expensive. Moreover, current detection technology makes it difficult or impossible to detect radioactive probes in real-time, thereby precluding study of kinetic processes.
CRF is a hypothalamic peptide hormone that plays an important role in coordinating autonomic, endocrine and behavioral stress responses in both the brain and the periphery. Specifically, CRF stimulates the synthesis and release of adrenocorticotrophic hormone (ACTH) from the pituitary gland. Beta-endorphin and other proopiomelanocortic (POMC)-derived peptides are synthesized and released from the cortex, cerebellum and possibly other sites in the body. The subsequent ACTH-induced release of adrenal glucocorticoids represents the final stage in the hypothalamic-pituitary-adrenal axis (HPA), which mediates the endocrine reponse to stress. CRF may also affect a variety of peripheral functions, including cardiovascular activity, inflammation, reproduction and integration of the immune system""s response to stress. Clinical studies have shown that CRF hypersecretion is associated with various diseases, such as major depression, anxiety-related illness, eating disorder, as well as inflammatory disorder. Low levels of CRF were found in Alzheimer""s disease, dementias, obesity, and many endocrine diseases. Several peptides, for example sauvagine, urocortin and urotensin have sequence and biological activity similar to that of CRF. Urocortin is a neuropeptide that may play a role in some CRF-mediated actions as well as appetite suppression and inflammation. Sauvagine regulates blood pressure, plasma hormone concentrations, and neuronal and gastric activity.
Corticotropin releasing factor (CRF) and related peptides all bind to CRF receptors that belong to the superfamily of G protein-coupled receptors, which includes calcitonin and calcitonin-like receptors. CRF related peptides are particularly desirable peptides to label and use to monitor cell receptors, as these peptides exhibit multiple biological roles and their receptors are located in a variety of tissues. For example, the CRF receptors fall into two distinct classes termed CRF1 and CRF2 receptors. The CRF2 receptor exists as three splice variants of the same gene that have been designated CRF2a, CRF2b and CRF2g. The pharmacology and localization of all CRF receptor proteins in the brain has been well established. The CRF1 receptor subtype is localized primarily to cortical and cerebellar regions of the brain, while the CRF2a receptor is localized to subcortical regions. The CRF2b receptor is primarily localized in the brain to cerebral arterioles and to choroid plexus heart skeletal muscle. The CRF2g receptor has most recently been identified in human amygdala.
There exists the need for CRF receptor-binding peptides that are chemically linked with detectable labels that are easily detected, yet do not decrease the biological activity of the peptide. Such labeled peptides are generally useful in the study of CRF related peptides and peptide binding receptors and may lead to the discovery of novel agents for treatment of depression, anxiety and other CRF related illnesses.
The present invention provides a compound containing a CRF related peptide and a light-emitting moiety that is both biologically active and optically detectable. The peptide is chemically attached to the light-emitting moiety at an amino acid position that is not involved in binding to the peptide receptor. In this way, the peptide""s affinity for the binding site is not significantly decreased, and the compound retains high biological activity. Furthermore, the compound can be easily detected using standard optical means.
In general, in one aspect, the invention provides a biologically active compound of the formula: 
where R1 is a light-emitting moiety, R2 is a CRF-related peptide and fragment, derivative or analog thereof and L is a linker moiety, which may be present or absent. The peptide is linked at a first amino acid position to (Cxe2x80x94X) which, in turn, is selected from the group including Cxe2x95x90O, Cxe2x95x90S, CH(OH), Cxe2x95x90Cxe2x95x90O, Cxe2x95x90NH, CH2, CH(OR), CH(NR), CH(R), CR3R4, and C(OR3)OR4 where R, R3, and R4 are alkyl moieties or substituted alkyl moieties. Optionally the compound may include a linker moiety between the peptide and the Cxe2x80x94X binding group. Preferably, the compound exhibits substantial biological activity in the presence of receptors having affinities for CRF-related peptides. The compound may also be in the form of a pharmaceutically acceptable salt or complex thereof. Preferably, the N-terminus of said CRF-related peptide is attached to (Cxe2x80x94X), either directly or through a linker moiety.
In preferred embodiments, the CRF related peptide can be any peptide that shares sufficient homology or activity with CRF (SEQ ID NO:1). In particularly preferred embodiments, the CRF related peptide is any one of sauvagine, urocortin, urotensin or CRF. Sauvagine includes the amino acid sequence Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gln-Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Thr-Ile (Sequence ID NO.2). Urocortin includes the amino acid sequence Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val (SEQ ID NO:3). Alternatively, the CRF related peptide could be a modified CRF or CRF related peptide that contains various substitutions, deletions or modified amino acids. The peptide [Nle 21,38]CRF contains norleucine at positions 21 and 38 and includes the amino acid sequence Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Ile (SEQ ID NO:4). It is preferred that the first residue of the peptide is attached to the (Cxe2x80x94X) moiety and is preferably chemically bound to the (Cxe2x80x94X) moiety through the N-terminal amino acid. In still other preferred embodiments, the (Cxe2x80x94X) bond is either Cxe2x95x90O or Cxe2x95x90S. In another preferred embodiment, the peptide may be amidated at the C-terminus.
In other preferred embodiments, the light-emitting moiety (R1) is selected from the group including 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, fluorescein, FITC, Texas red, phycoerythrin, rhodamine, carboxytetramethylrhodamine, 4xe2x80x26-diamidino-2-phenylindole, indopyras dyes, Cascade blue, coumarins, nitrobenzo-2-oxa-diazole (NBD), Lucifer Yellow, propidium iodide, CY3, CY5, CY9, dinitrophenol (DNP), lanthanide cryptates, lanthanide chelates, non-fluorescent dialdehydes (OPA, NDA, ADA, ATTOTAG reagents from Molecular Probes) which react with primary amines (N-term Lys) in the presence of a nucleophile (i.e. CNxe2x88x92) to form fluorescent isoindoles, dansyl dyes fluorescamine and dabcyl chloride, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, long lifetime dyes comprised of metal-ligand complexes (MLC) which consist of a metal center (Ru, Re, Os) and organic or inorganic ligands complexed to the metal such as [Ru(bpy)3]2+ and [Ru(bpy)2(dcbpy)], and the like and derivatives thereof. The light-emitting moiety can be attached to the peptide by reaction of a reactive side group (of the light-emitting moiety) with the N-terminal amino acid of the CRF related peptide. Suitable linking moieties include, by way of example only, indoacetamide, maleimide, isothiocyanate, succinimidyl ester, sulfonyl halide, aldehydes, glyoxal, hydrazine and derivatives thereof.
The above-identified compound is useful in the labeling of cell receptor sites, cell sorting, flow cytometry and performing fluoroimmunoassays. In another aspect, the invention provides a method for labeling a receptor having an affinity for a CRF related peptide by contacting the receptor with one or more of the compounds described above. Cell receptor sites, can be imaged by contacting candidate cell receptor sites with the compound of the invention, and then detecting the bound compounds as an indication of the cell receptor sites. Cell sorting can be performed by contacting a population of cells with compound and isolating cells bound to the compound. Flow cytometry can be performed by contacting a population of cells with the compound and detecting cells bearing receptors on their surfaces by detecting cells bound to the compound.
The invention has many advantages. In a general sense, peptide-containing compounds which retain their biological activity after being labeled with light-emitting moieties have a wide variety of biological applications. Such compounds can be used to identify, visualize, quantify, target and select receptors on cells and tissues both in vitro and in vivo. These compounds may be used in place of more conventionally labeled peptides, such a 125I radiolabeled peptides. Radiolabeled compounds are often toxic, environmentally hazardous, chemically unstable and have, by the nature of the radioactive decay rate, relatively short lifetimes. In contrast, fluorescently-labeled CRF related peptides are relatively safe and non-toxic, thereby allowing it to be synthesized and used without employing special laboratory procedures. Similarly, following use, fluorescent CRF related peptides may be easily disposed, whereas disposal of radioactive compounds is both time-consuming and costly. In addition, fluorescent markers for CRF related peptide receptors are stable and may be stored for extensive periods of time without undergoing considerable degradation.
Use of CRF related peptides in the labeled compound provides several additional advantages. As described above, CRF related peptides exhibit biological activity in organs such as the brain and heart skeletal muscle, and are involved in stress response in a variety of tissues. Therefore compounds containing CRF related peptides may be used as probes to investigate a number of different cell types. In addition, these peptides have a relatively simple structure (about 40 amino acids) and can be synthesized and isolated with standard, well-known techniques.
During typical experiments, fluorescent markers for CRF related receptors emit optical signals, and thus may be monitored by eye or with the aid of external optical detectors. In this way, the fluorescent peptides obviate the need for secondary detection steps sometimes used for radiolabeled compounds or incubation with secondary labeled compounds. Detection of optical radiation is, in general, relatively simple and cost-effective compared to detection of radioactive particles (e.g., xcex1-particles); conventional charge-coupled device (CCDs) or light-sensitive cameras can therefore be used without modification for this application.
In addition, because of their high optical emission rates and well-characterized emission cross sections, fluorescent markers attached to CRF related peptide receptors can be used for real-time, quantified imaging of a number of dynamic biological phenomena, such as kinetics associated with receptor-binding. The compounds can also be used for static processes, such as monitoring peptide distribution within a cell. CRF related peptide receptors marked with fluorescent peptides may also be used in flow cytometry, confocal microscopy, fluorescence polarization spectroscopy, and any other techniques exploiting the optical detection of fluorescence or photoluminescence.
Other advantages and features of the invention will become apparent from the following detailed description, and from the claims.
Definitions
xe2x80x9cCRF related peptidexe2x80x9d or xe2x80x9cCRF related compoundxe2x80x9d: xe2x80x9cCRF related peptidexe2x80x9d or xe2x80x9cCRF related compoundxe2x80x9d refers to any peptide that binds to the family of G protein-coupled receptors, for example CRF1, or CRF2 receptors, (including CRF2a, CRF2b and CRF2g, and CRF receptors located on rat cortex membranes). For example, any protein with substantial sequence homology and similar biological activity to CRF related peptides, as determined by one of ordinary skill in the art, would be considered to qualify as a CRF related peptide. xe2x80x9cCRF related peptidexe2x80x9d or xe2x80x9cCRF related compoundxe2x80x9d is meant to include fragments of CRF related peptides, derivatives or analogs thereof. Some examples include, sauvagine, urocortin, urotensin and CRF as described in the present application. CRF related peptides may be peptides whose sequences differ from wild-type sequences by only conservative amino acid substitutions. For example, one amino acid may be substituted for another with similar characteristics (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the peptide""s biological activity. Alternatively the CRF related peptide may contain modified amino acids such as norleucine in [Nle 21, 38]CRF. Other useful modifications include those which increase the peptide""s stability. The peptide may contain, for example, one or more non-peptide bonds (which replace a corresponding peptide bond) or D-amino acids in the peptide sequence. Additionally, the C-terminus carboxylic acid group may be modified to increase peptide stability. For example, as described above, the C-terminus may be amidated or otherwise derivatived to reduce the peptide susceptibility to degradation. For example, in the present invention, all peptides are modified by a carboxy-terminal NH2 group that protects the peptide from degradation by the enzyme carboxypeptidase.
xe2x80x9cFluorescent peptidexe2x80x9d or xe2x80x9ccompoundxe2x80x9d: As referred to herein, xe2x80x9cfluorescent peptidexe2x80x9d or xe2x80x9ccompoundxe2x80x9d refers to a peptide-based compound that has been labeled with a light emitting moiety. The fluorescent peptide has the desirable characteristics of preserving the biological activity of the peptide-based compound, for example in receptor binding, and providing a detectable signal that can be measured using standard optical means.
xe2x80x9cLight emitting moleculexe2x80x9d: xe2x80x9cLight emitting molecule,xe2x80x9d as used herein, refers to a molecule capable of emitting light of any detectable wavelength that is not attached to a peptide of the present invention.
xe2x80x9cLight emitting moietyxe2x80x9d: xe2x80x9cLight emitting moietyxe2x80x9d is used to refer to a light emitting molecule (e.g., a fluorescent dye) that has been attached by any of a variety of means, as described below, to peptide-based moiety. Attachment to the peptide-based moiety is carried out so that the biological activity of the peptide-based moiety is maintained. The light emitting moiety provides a detectable signal of a particular wavelength. In general, the signal provided by light emitting moieties may be detected by a variety of techniques including conventional microscopy methods, including fluorescence or confocal microscopy, atomic force microscopy, fluorescence polarization spectroscopy and fluorimetry. Particularly preferred light emitting moieties are described in more detail below.
xe2x80x9cPeptide moietyxe2x80x9d: xe2x80x9cPeptide moietyxe2x80x9d, as used herein, refers to any peptide composed any sequence of natural (i.e., found in nature) and/or custom amino acids. By xe2x80x9ccustom amino acidxe2x80x9d is meant any amino acid that can not be found in nature, but can be synthesized in a laboratory. Such amino acids are often chemically modified amino acids. It is well known that natural amino acids may also be synthesized. Particularly preferred peptide moieties of the present invention include peptide moieties that bind G protein-coupled receptors. Most particularly preferred are peptide moieties that bind CRF receptors (CRF1 and CRF2).
xe2x80x9cLinker moietyxe2x80x9d or xe2x80x9clinkerxe2x80x9d: A xe2x80x9clinker moietyxe2x80x9d or xe2x80x9clinkerxe2x80x9d is any moiety of the compound located between the peptide and the label or at any other position which provides greater three dimensional separation between the label and the peptide. One particularly preferred linker moiety used in the present invention is based on xcex3-aminobutyric acid. Other moieties that may be used as linkers in the present invention include those derived from glycine, beta-alanine, aminopentanoic acid, aminohexanoic acid, aminohepanoic acid, aminooctanoic acid, aminononanoic acid, aminodecanoic acid, aminoundecanoic acid, and aminododecanoic acid. Each of these moieties include an amino and a carboxylic acid functionality and so may be incorporated into the compound using a peptide bond.
xe2x80x9cBiologically active compoundxe2x80x9d or xe2x80x9cbiologically active peptidexe2x80x9d: xe2x80x9cBiologically active compoundxe2x80x9d or xe2x80x9cbiologically active peptidexe2x80x9d, as used herein, refers to the fluorescently labeled peptide of the invention represented in the formula described below and in FIG. 1. Any biologically active compound of the present invention is substantially biologically active.
xe2x80x9cSubstantially biologically activexe2x80x9d: In all cases, by xe2x80x9csubstantially biologically activexe2x80x9d is meant that the compound binds to a receptor having an affinity IC50 or Ki value for the compound which is no more than 100 times, preferably no more than 15 times, more preferably no more than 10 times and most preferably equal to or less than that of the corresponding unlabeled peptide. Most preferably, an affinity IC50 or Ki value for the compound is no more than 10 nM. Receptor affinity in this case can be determined using known methods, such as methods involving competitive binding of radioactively labeled peptides or by using known methods of fluorescence polarization or other known fluorescence technique for measuring the Kd for the receptor/peptide interaction.
xe2x80x9cLowxe2x80x9d or xe2x80x9cnoxe2x80x9d biological activity or xe2x80x9cbiologically inactivexe2x80x9d: By xe2x80x9clowxe2x80x9d or xe2x80x9cnoxe2x80x9d biological activity or xe2x80x9cbiologically inactivexe2x80x9d is meant biological activities less than 1.0% of the biological activity of R2xe2x80x94H in the presence of a receptor having affinity for CRF related peptides.